Whole blood separation method and devices using the same

ABSTRACT

The present invention relates to a method and to a device for separating plasma from whole blood. The method and device utilize a permeable non-glass fiber matrix containing a polyol which is capable of clumping red blood cells. The matrix, in the absence of such a polyol, would otherwise be porous to red blood cells. The polyol-containing matrix has a first surface and a second surface such that a whole blood sample which is applied to the first surface flows directionally toward the second surface. Plasma separated from whole blood becomes available at the second surface of the matrix and can be tested for the presence of a particular analyte, such as glucose or fructosamine, as provided by multi-layer test devices of the present invention.

FIELD OF THE INVENTION

The present invention relates generally to the chemical analysis ofanalytes present in whole blood and more specifically, to a method andto a device for separating plasma or serum from whole blood, thusproviding a convenient and accurate means for such chemical analysis.

BACKGROUND INFORMATION

Presently, numerous test devices are available for the analysis of bodyfluids in order to determine the presence or concentration of aparticular analyte. For example, tests are available for detectingglucose, fructosamine, albumin, calcium, urea, uric acid, bilirubin,cholesterol, and other soluble analytes present in whole blood or thefluid part of blood, namely the plasma or serum, after whole blood hasbeen separated.

Many of these test devices utilize chromogenic or other visual responsesto indicate the presence or absence, or the concentration, of an analytebeing detected. Cellular components of whole blood, and in particularthe red blood cells, have a deep red color which substantiallyinterferes with chromogenic or other visual tests. Therefore, thehighly-colored red blood cells as well as other interfering substancespresent in blood including hemoglobin and white blood cells areseparated from the plasma or serum before a blood sample is assayed fora particular analyte.

Conventionally, the plasma or serum is separated from the cellularmaterial of whole blood by centrifugation. The cellular materialcollects at the bottom of the centrifuge tube and the supernatant plasmaor serum is decanted and tested for a particular analyte.Centrifugation, however, is time consuming, involves extra manipulativesteps and requires equipment that is generally not present outside ofthe clinical laboratory. Thus, reliance on centrifugation makes fieldtesting, such as testing at the doctor's office or at the patient'shome, difficult.

Certain methods other than centrifugation have been developed toseparate the cellular components of whole blood from plasma or serum.Some of the earlier methods, such as that described in U.S. Pat. No.4,543,338 to Chen, involve the use of a carrier membrane impregnatedwith a test reagent and coated with a semipermeable membrane. Thesemipermeable membrane effectively acts as a means for filtering outcells or large molecules, such as hemoglobin, but allows the passage ofsmaller molecules and ions which then contact the testing reagentsimpregnated in the bibulous matrix. These methods, however, typicallyrequire an extra manipulative step, such as rinsing with water or wipingoff the test device so as to remove cellular material retained on thesemipermeable membrane. Such techniques can be cumbersome and laborious.Moreover, if the red blood cells are not completely removed or rinsedfrom the semipermeable barrier, interference with the assay remains aproblem.

Another method for separating whole blood is described in U.S. Pat. No.4,477,575 to Vogel et al. which describes separating plasma or serumfrom whole blood using a layer of glass fibers having a defined averagediameter and density. As well, Baumgardner et al. in U.S. Pat. No.5,186,843 describe the use of glass fibers in a single separation layer.Blood separation devices utilizing glass fiber filters, however, tend toseparate serum at a relatively slow speed and tend to retain significantquantities of serum or plasma in the interstices of the glass fibermatrix.

Alternative approaches to blood separation involve incorporatingagglutinating reagents or other separation reagents in a matrix. Forexample, Aunet et al. in U.S. Pat. No. 4,933,092, Daubney et al. inpublished Canadian Patent Application No. 2,104,976, and Limbach inEuropean Patent Application 0 194 502 all describe the use of polymericagglutinating agents, such as cationic polymers, which, for example, inDaubney and in Limbach are combined with additional agglutinatingagents, such as lectins. As well, Barkes et al. in European PatentApplication No. 0 436 897, describe the use of lectins and thrombinincorporated into a suitable carrier matrix. However, such matricesincorporated with these types of agglutinating agents exhibit problemssimilar to those associated with glass fiber matrices. For example, theseparation may occur at a relatively slow speed and the amount of plasmaor serum separated may be limited to 50% of the absorption volume of thematrix, often requiring the use of external pressure, such as inEuropean Patent Application 0 194 502, in order to obtain the maximumefficiency and quantity of plasma or serum.

Other separating agents, such as water-soluble salts, amino acids,carbohydrates and large polymers, such as polyethylene glycol, polyvinylalcohol and the like, have been incorporated into single matrix teststrips. Fetter in U.S. Pat. Nos. 3,552,925 and 3,552,928 describes atest device having a bibulous matrix impregnated with an inorganic saltor amino acid at a first region on the matrix and test reagentsimpregnated at an adjacent second region. While the salts or amino acidsused in this process can separate the cellular components from the wholeblood, they also introduce contaminating ions or molecules into theplasma or serum and precipitate a portion of the soluble plasma or serumconstituents, thus rendering a quantitative assay for the solubleconstituent unreliable. Rapkin et al. in U.S. Pat. No. 4,678,757describe the use of carbohydrates, such as mannitol, impregnated orcoated onto a carrier, preferably coated onto an impermeable carrier.The described device, whether having a permeable or impermeable carrier,however, only provides for capillary and longitudinal transport of theblood. Therefore, the blood separation matrices of Rapkin et al. are notdescribed as being useful in test devices that operate primarily bygravitational force, as do many of the multi-layer test devicescurrently used by doctors or patients. While Kiser et al. in U.S. Pat.No. 5,306,623 describe separation matrices which can operate by wickinggravity flow, Kiser et al. use large polymeric separating reagents, suchas polyethylene glycol, polystyrene sulfonic acid, hydroxypropylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylicacid. Upon application of a whole blood sample, such large polymerscontained within a matrix would be solubilized, potentially blocking thepores of the matrix and most certainly rendering the sample moreviscous, thereby slowing the separation process and decreasing the yieldof plasma.

Based on the shortcomings of these methods, there exists a need for adevice that provides rapid and efficient methods for the separation ofplasma or serum from whole blood. In particular, there is an increasingawareness of the importance of, and accordingly a need for, being ableto carry out diagnostic assays at the doctor's office or, better yet, athome. Therefore, there exists a need to minimize any extra manipulativesteps, such as the rinsing or wiping of test devices or the applicationof external pressure. Moreover, there is a need for rapid separationwhich is reliable and does not contain interfering substances. Thepresent invention satisfies these needs and provides related advantagesas well.

SUMMARY OF THE INVENTION

The present invention relates to a method and to a device for separatingplasma from whole blood. The method and device utilize a permeablenon-glass fiber matrix containing a polyol which is capable of clumpingred blood cells. The matrix, in the absence of such a polyol, wouldotherwise be porous to red blood cells. The polyol-containing matrix hasa first surface and a second surface such that a whole blood samplewhich is applied to the first surface flows directionally toward thesecond surface. Plasma separated from whole blood becomes available atthe second surface of the matrix and can be tested for the presence of aparticular analyte, such as glucose or fructosamine, as provided bymulti-layer test devices of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one embodiment of a multi-layer fructosamine test devicewhich can be used for separating plasma from a whole blood sample andmeasuring the concentration of fructosamine.

FIG. 2 exemplifies one embodiment of a glucose test device which isuseful for separating plasma from whole blood and measuring theconcentration of glucose.

DETAILED DESCRIPTION OF THE INVENTION

There is an increasing awareness of the importance of being able tocarry out diagnostic assays at the doctor's office or, better yet, athome. For example, a diabetic's blood glucose level fluctuatessignificantly throughout a given day, being influenced by diet,activity, and treatment. Depending on the nature and severity of theindividual case, some diabetic patients measure their blood glucoselevels up to seven times a day. Clearly, the results of these testsshould be available to the patient immediately.

Because of the frequent fluctuation of glucose levels in a given day,tests which are independent of a patient's diet, activity, and/ortreatment and which provide longer term indications of blood glucoselevels have been developed. These tests measure the concentration ofglycated proteins or "frucosamines." Proteins, such as those present inwhole blood, plasma, serum and other biological fluids react withglucose, under non-enzymatic conditions, to produce glycated proteins.The extent of the reaction is directly dependent upon the glucoseconcentration of the blood. Measurement of serum or plasma fructosaminelevels is useful for monitoring diabetic control because fructosamineconcentrations in serum or plasma reflect an average of blood glucoselevel over approximately a half month period.

Scandinavian investigators recently showed that doctors and patients whowere made aware of their glycated protein test results had betterglycemic control than those who were unaware of such results. Moreover,it is now believed that glycated proteins can be the causative agents ofcomplications associated with diabetes, which include retinopathy,nephropathy, neuropathy and cardiovascular disease. Therefore, any delayin information transfer, such as a doctor's delay in reporting clinicaltest results to a patient, decreases the value of the test result.Again, this emphasizes the importance in being able to performdiagnostic assays at the doctor's office or at home.

For an assay to be useful in the doctor's office or home, the testshould be relatively free of sensitivity to small changes in theconditions under which the assay is carried out and the measurementsshould be accurate and reliable. Equally as important, if not more so,the assay must have a simple and convenient protocol which does notinvolve extra manipulative steps. To enhance the simplicity andconvenience of such tests, the preferable body fluid for testing suchanalytes as glucose and fructosamine is whole blood which can simply betaken from a finger or earlobe puncture. Such simple and rapiddeterminations of an analyte in blood is especially desirable in thecase of an emergency.

As described above, the simplicity and accuracy of such tests depends toa large extent on the whole blood separation layer contained within thetest device and the ability of the blood separation matrix to provideuncontaminated plasma or serum. The present invention provides a methodand device, namely a blood separation matrix, which is capable of suchsimple and accurate blood separation. Of particular importance, thematrix contains a polyol which causes red blood cells contained in wholeblood to clump. The clumped red blood cells either can be retained inthe matrix or can be filtered by a filter material. The method andmatrix can be used in various test devices which analyze whole blood fora particular analyte, such as, the fructosamine and glucose test devicesprovided by the present invention.

As used herein, the term "plasma" means the substantially colorlessfluid obtained from a whole blood sample after red blood cells have beenremoved by the separation process and device of the present invention.Because plasma is serum plus the clotting protein fibrinogen, the term"plasma" is used broadly herein to include both plasma and serum.

In order to obtain plasma from whole blood, the present inventionprovides a permeable non-glass fiber matrix containing a polyol which iscapable of clumping red blood cells. The matrix is porous to red bloodcells in the absence of such a polyol. The matrix has a first surfacefor sample application and a second surface where plasma is received orbecomes available. If desired, the matrix additionally can contain apolycationic polymer. Also useful, though not required, is a permeablefilter material or membrane supporting the separation matrix whichserves as a final filter to red blood cells and/or provides a reagentlayer for effecting an assay. Each of these components of the invention,as well as test devices which use the blood separation method and matrixof the present invention are disclosed in detail.

MATRIX

The separation matrix of the present invention is a permeable matrixwhich does not contain glass fibers and, therefore, is termed "apermeable non-glass fiber matrix." The term "permeable" meansliquid-permeable, such as permeable to plasma, as well as permeable orporous to red blood cells when the matrix is provided in the absence ofa polyol. As used herein, the phrase "matrix being porous to red bloodcells in the absence of a polyol" means that without the polyolcontained in or on the matrix the red blood cells would simply passthrough the matrix, virtually immediately. In the absence of the polyol,red blood cells are not retained, by filtration or otherwise, in thematrix.

The polyol contained within or on the matrix chemically reacts with thewhole blood sample so as to clump the red blood cells. As used herein,"clump" or "clumping" means the collection into a mass or group, redblood cells distributed in a whole blood sample. While not wishing to bebound by any theory or mechanism, the clumping can be the result ofagglutination, coagulation, or the like, or some other chemicalinteraction between the polyol and the red blood cells. Thus, thepresent invention is not strictly a filtration process, for example,based on the pore size of the matrix, as described, for example, in U.S.Pat. No. 4,543,338 to Chen, or as used in a glass fiber prefilter, suchas that described in U.S. Pat. No. 4,477,575 to Vogel et al. Rather, itis the presence of a polyol which provides the matrix with its abilityto separate plasma from whole blood by clumping the red blood cells.

Surprisingly, the clumping of the red blood cells by the polyol does notsubstantially block the flow of the whole blood sample or plasma throughthe matrix. Thus, sufficient amounts of plasma become available at thesecond surface of the matrix. Sufficient quantities of plasma canrapidly be obtained for the specific applications exemplified herein,namely analyzing the concentration of frucotosamine or glucose in dropsof whole blood. The present invention can be used with otherapplications and diagnostic assays as well, including ones which use alarger volume of blood or which require more plasma. With as small as a3/16" diameter circle of a polyol-containing matrix of the presentinvention, as much as 10 μl of plasma can be obtained from a drop ofblood. Therefore, a matrix of the present invention can be used forseparating larger volumes of blood than a drop of blood. Accordingly,the matrix can be used in diagnostic applications which analyze largerquantities of blood, such as for example, some known cholesterol tests.

A useful permeable matrix can be a woven or non-woven material and canbe an absorbent or a non-absorbent material which may or may not behydrophilic. Especially suitable materials for the matrix include, forexample, woven or non-woven, absorbent or non-absorbent, nylon, rayon,cotton, and polyester. In one embodiment of the invention, the matrix isa non-woven, non-absorbent polyester. The polyester is preferably apoly(paraphenylene terephthalate), such as that used in a preferredpolyester sold as Sontara® (DuPont, Inc., Wilmington, Del.). Anotherpreferred matrix is the woven, absorbent nylon Tetex® 3-3710 (Tetko,Inc., Lancaster, N.Y.).

Depending upon the porosity or other properties of the matrix, theclumped red blood cells either are retained in the matrix or arefiltered out by the filter material as described below. Some of theabove-described matrix materials, such as the non-woven, non-absorbentpolyesters, do not have "pores" in the traditional sense, i.e., that canbe measured, for example, by pore size (microns). In the absence of apolyol of the present invention such materials essentially have no limitas the porosity and are porous to red blood cells, which have an averagesize of 5 μm. With such macroporous materials, if the polyol is notpresent the red blood cells pass through the matrix almost immediately.For those matrix materials which can be characterized based on poresize, the matrices used in the present invention can have a pore sizegenerally of from about 2 μm to about 10 μm. Such pores sizes can beuseful for retaining the clumped red blood cells. Depending upon theporosity, thickness, which is generally 200 to 1100 μm, and otherproperties of the matrix, such as absorbency, the clumped red bloodcells are either retained in the matrix or captured in a final filtermaterial as described below.

The polyol-containing matrix has a first surface for sample applicationand a second surface where plasma is received or becomes available fortesting or additional separation. Generally, the first and secondsurfaces are presented as opposite sides of the matrix. The whole bloodsample flows in a direction from the first surface toward the secondsurface, under conditions which provide such directional flow, such as,gravitation, vacuum, or external pressure. To enhance the simplicity ofthe method, if desired, separation can be performed by gravity alone.Preferably, the separation matrix provides for flow in a verticaldirection, preferably by gravitation.

Rapkin et al. in U.S. Pat. No. 4,678,757, describe the use ofcarbohydrates, such as mannitol, impregnated or coated onto a carrier,preferably coated onto an impermeable carrier. However, the describeddevice of Rapkin et al., whether having a permeable or impermeablecarrier, only provides for capillary and lateral transport of the blood.There can be divergent contact times provided by vertical flow, arelatively short period of contact, versus lateral flow, a slow processwhich can involve continuous interaction between a whole blood sampleand a matrix. Because of these variable contact times, it is notpredictable that what works by lateral flow would similarly work undervertical flow. Unexpectedly, with the present invention, even withmatrices which are porous to red blood cells in the absence of a polyol,a matrix containing a polyol can effectively separate plasma from wholeblood even when the blood sample flows vertically through the matrix.

POLYOL

The separation method and device include a permeable non-glass fibermatrix containing a polyol. As used herein, the terms "matrix containinga polyol" and "polyol-containing matrix" mean that the polyol isseparately added to the matrix and is not a component originally foundin the composition or make up of the matrix, such as cellulose filterpaper. Further, "matrix containing a polyol" means a polyol can beimpregnated into the matrix or coated into or onto the matrix orcovalently or non-covalently bound to the matrix. In a preferredembodiment, the polyol is impregnated into the matrix.

As used herein, the term "polyol" means a polyhydroxy alcohol which isan alkyl or aromatic containing more than one hydroxyl group. The term"poly" as used in "polyol" does not infer that the alkyl or aromaticcompound is a large polymer made up of repeating monomeric units, but,instead, means that more than one hydroxyl group is present in thecompound. As discussed more fully below, with the exception ofpolysaccharides, the polyols used in the present invention are simplesugars or sugar alcohols, oligosaccharides, or other naturally ornon-naturally occurring non-polymeric alkyl or aromatic compounds.Therefore, the term "polyol" encompasses sugars, alcohol derivatives ofsugars, herein termed "sugar alcohols," and other naturally ornon-naturally occurring non-polymeric polyols.

As used herein, "sugar" includes monosaccharides, oligosaccharides, andpolysaccharides. A monosaccharide is a simple sugar which is as alinear, branched, or cyclic polyhydroxy alcohol containing either analdehyde or a ketone group. Exemplary monosaccharides include, but arenot limited to, mannose, glucose, talose, galactose, xylose, arabinose,lyxose, ribose and fructose. An oligosaccharide is a linear or branchedcarbohydrate that consists from two to ten monosaccharide units joinedby means of glycosidic bonds. Oligosaccharides which can be used in thepresent invention include, but are not limited to disaccharides such assucrose, trehalose, lactose and maltose. Examples of largeroligosaccharides which can be used in the invention include thecyclodextrins, such as alpha-cyclohexylamylose, beta-cycloheptaamylose,and gamma-cyclooctoamylose, as well as other oligosaccharides well knownin the art. A polysaccharide is any linear or branched polymer havingmore than ten monosaccharides linked together by glycosidic bonds.Exemplary polysaccharides include, but are not limited to, ficoll,polysucrose, and hydroxyethyl starch.

Encompassed within "sugar" are those sugars which are naturallyoccurring as well as those which are known but which have not yet beenidentified as occurring naturally in plants or animals. For example,there are five known naturally occurring aldohexoses, includingD-glucose, D-mannose, D-talose, D-galactose, and L-galactose. However,the aldohexose structure has four chiral carbons and thus, sixteenpossible stereoisomers, all of which are known, although only the fivelisted above have been identified as occurring naturally in plants oranimals. Thus, "sugar" encompasses enantiomers in either the D or Lforms of a sugar as well as racemic mixtures thereof.

A polyol of the present invention also can be a "sugar alcohol." A"sugar alcohol" is an alcohol derivative of a mono- or anoligosaccharide which is generally formed by reduction of the aldehydeor ketone moiety on the mono- or oligosaccharide. Exemplary sugaralcohols include, but are not limited to, mannitol, sorbitol, arabitol,inositol, galactitol, erythritol, and threitol. Also included within thedefinition of "sugar alcohol" are the alcohol derivatives of those mono-and oligosaccharides described above.

Where chiral carbons are present in the sugar alcohol, the sugar alcoholmay be in the D or L form, such as D-threitol or L-threitol, or in aracemic mixture of both the D and L forms. The sugar alcohol can, butdoes not have to, be naturally occurring. That is, the sugar alcohol canbe a derivative of a known, naturally occurring sugar, or,alternatively, it can have a D or L configuration known to exist but notnecessarily identified as occurring in nature. The sugar alcohol alsocan be a sugar which is found naturally in its reduced alcohol form orit can be an alcohol derivative of a sugar which derivative is not knownto exist in nature.

In addition to sugar or sugar alcohols, the polyol can be anon-polymeric naturally occurring or non-naturally occurring polyol,which includes linear, branched, or cyclic alkyl or aromatic compoundscontaining more than one hydroxyl group. As used herein the term"non-polymeric" means the alkyl or aromatic compounds are not polymers.Polymers are defined as high molecular weight compounds consisting oflong chains that may be open, closed, linear, branched, or crosslinked,which chains are composed of repeating units, called monomers, which maybe either identical or different. As used herein, those polyols whichare "naturally occurring" are ones which occur in nature and those whichare "non-naturally occurring" are not found in nature. Generally, thesenaturally occurring or non-naturally occurring alkyl or aromaticcompounds range in size from three to twenty carbons (C₃ to C₂₀), andmore preferably, from three to ten carbons (C₃ to C₁₀). Examples of suchnaturally occurring, non-polymeric polyols are glycerol, a three-carbontrihydroxy alcohol that occurs in many lipids, and quinic acid, a1,3,4,5-Tetrahydroxycyclohexanecarboxylic acid, which acid can be in thesalt form. Examples of non-naturally occurring, non-polymeric polyolsinclude pentaerythritol and dipentaerythritol.

Kiser et al. in U.S. Pat. No. 5,306,623 describe the use of largepolymeric separating reagents, such as polyethylene glycol, polystyrenesulfonic acid, hydroxypropyl cellulose, polyvinyl alcohol,polyvinylpyrrolidone, and polyacrylic acid. Upon application of a wholeblood sample, such large polymers contained within a matrix would besolubilized, potentially blocking the pores of the matrix and mostcertainly rendering the sample more viscous, thereby slowing theseparation process and decreasing the yield of plasma. Therefore, withthe exception of the above-described polysaccharides, the invention doesnot involve the use of large alkyl polymers as the primary separatingagent. The matrix described in U.S. Pat. No. 5,306,623 is different fromthe instant invention in a number of other aspects as well. Forinstance, the Examples given in U.S. Pat. No. 5,306,623 involve the useof matrices which have a very small pore size, less than 1 μm, whichwould act as a filter to red blood cells in the absence of the disclosedpolymers. As discussed above, the present invention is not strictly afiltration process, rather it involves the use of polyols which clumpred blood cells and in the absence of such polyols the matrix would beporous to red blood cells. Moreover, the matrices taught by Kiser etal., in addition to a polymer, also contain the test reagents, whichreagents may substantially influence the separation and test results.The separation matrix of the present invention does not contain the testreagents. The test reagents, as disclosed in greater detail below, areeither on the filter material or additional test reagent layers and thelike.

In one embodiment, to apply the polyol to the matrix, the polyol cansimply be dissolved in an aqueous solution generally, at a concentrationof about 20% when used alone, and at about 10% concentration whencombined with a polycationic polymer, which is generally present in aconcentration of about 0.5% to 5% as discussed more fully below. Ifdesired, multiple layers of matrices containing polyol at lowerconcentrations, such as four layers of matrix containing 5% polyol, alsocan be used. The polyol and, if present, the polycationic polymer canalternatively be dissolved in physiological saline (0.85% NaCl),phosphate buffered saline (PBS), an organic solvent, or the like.

POLYCATIONIC POLYMER

In addition to the polyol, a polycationic polymer can, but does not haveto, be added to the matrix. Similar to the addition of a polyol to thematrix, the polycationic polymer can also be physically impregnated,coated into or onto, or covalently or non-covalently bound to thematrix. The polycationic polymer is also useful for clumping, as well asstabilizing clumped, red blood cells, the latter of which is describedfor example, in the published Canadian Patent Application No. 2,104,976to Daubney et al., which is incorporated herein by reference.

The polycationic polymer component can be any polymer having more thanone cationic site and are generally based on monomers which contain anamine group. Suitable polycationic polymers include, for example,hexadimethrine bromide, trimethylenehexamethylenediammoniumbromide,polylysine, polyallylamine, polyarginine,poly(N,N-dimethylaminoethylmethacrylate, copolymers ofN,N-dimethylaminoethylmethacrylate and methylmethacrylate,polyethyleneimine, poly(diallyldimethylammonium chloride),poly(1,1-dimethyl-3,5-dimethylenepiperidinium chloride), and mixturesthereof. The polymerized positively charged amino acids, such aspolylysine, can have the amino acids in either the D or L forms, such aspoly-L-lysine or poly-D-lysine, or a racemic mixture thereof, such aspoly-D,L-lysine.

As described above, in one embodiment, to apply the cationic polymer tothe matrix, the polymer can be dissolved in an a solution such as water,physiological saline, PBS, an organic solvent, or the like, and thematrix then dipped into the polymer containing solution. Generally, thepolymer is in a concentration of about 0.5% to 5%. Where both polyol andpolymer are contained in the matrix, the order of adding polyol andpolymer to the matrix is irrelevant. For example, polyol and polymer canbe simultaneously or sequentially dissolved in such aqueous solutions orsolvents as those described above and both polyol and polymersimultaneously applied to the matrix, as described in the Examplesbelow. Alternatively, polyol and polymer can be applied to the matrixsequentially in any order.

Non-hemolytic detergents, such as Pluronic (Pragmatics, Inc., Elkhart,Ind.), can be added to the aqueous solutions or solvents describedabove, generally at a concentration of 0.01% to 0.1%. Such detergentshelp maximize impregnation of a polyol into the matrix, therebyimproving the flow rate of the whole blood sample and the plasma. Otheroptional agents which can further enhance the flow rate, include, forexample, polyvinylpyrrolidone or similar polymers and other fillerswhich give the matrix and the below described filter material stiffness.

FILTER

Though not required, a filter material can be used in combination withthe matrix of the present invention. Suitable filter materials include,for example, nylon, cellulose acetate, polysulfone, synthetic fibers,and polycarbonate. The filter can, but does not have to, be a membrane.Illustrative filters and membranes include, for example, BTS polysulfonemembrane (Memtek, Inc., San Diego, Calif.), Ahlstrom synthetic fibersheets, such as 94-30 A (Ahlstrom Filtration, Inc., Mt. Holly Spring,Pa.), Biodyne A® nylon membrane (Pall Corp., East Hills, N.Y.),Ultrabind 450 (Gelman, Ann Arbor, Mich.), and Nucleopore® polycarbonate(Costar, Corp., Cambridge, Mass.).

The need for any additional filter material depends to a large extent onthe porosity, thickness, absorbency or other properties of the matrix.For example, the clumped red blood cells, depending upon the aboveproperties of the matrix, can be retained in the matrix. Alternatively,or in addition thereto, a final filter material can be used to captureor retain any additional clumps of red blood cells. Where present, thefilter material can generally have a porosity of up to about 12 μm andpreferably will have a pore size of less than 10 μm, and more preferably5 μm or less.

A filter material can be placed underneath the polyol-containingseparation matrix, thereby supporting the matrix. Because the filter ormembrane is at the second surface of the matrix where the plasma becomesavailable, the filter material can also serve as a reagent layer. Thefilter material can contain at least one chemical reagent fordetermining the presence of an analyte in the plasma. Determining thepresence of an analyte can be a qualitative or quantitativedetermination.

In preferred embodiments of the invention, the blood separation methodand device comprise a non-woven, non-absorbent polyester matriximpregnated with mannitol and either a nylon or polysulfone membranebelow the matrix. Preferably, the matrix additionally containshexadimethrine bromide. The membrane can additionally contain at leastone chemical reagent for analyzing the concentration of an analyte suchas glucose present in a whole blood sample.

In one embodiment of the invention, provided in FIG. 2 and Example 2,the filter membrane contains test reagents for determining glucoseconcentration in whole blood. Referring to FIG. 2, the glucose testdevice of the present invention has a polyol-containing blood separationmatrix 15 which can be held in position by a mask 14 and a plasticsupport member 16, below the latter of which is a membrane 17 containingglucose test reagents, held in position by a plastic support member 18.

Chemical reagents for determining the presence or absence or theconcentration of various analytes, such as glucose or fructosamine, arewell known in the art. For example, such test reagents which produce asignal in response to glucose typically involve a glucose oxidase enzymereaction. Glucose and glucose oxidase enzyme react to produce hydrogenperoxide. A peroxidase, such as horse radish peroxidase, and a redoxindicator, such as o-tolidine, o-dianisidine,3,3,5,5-tetramethylbenzidine (TMB), 4-aminoantipyrine, and others wellknown in the art, can be oxidized in the presence of hydrogen peroxideto produce a colored product. Such reagents for determining glucosepresence and concentration are disclosed, for example, in EuropeanPatent Application 0388782 to Chen, and U.S. Pat. No. 5,304,468 toPhillips et al., both of which are incorporated herein by reference.

As described above, though not required, the filter material can serveas a final filter in the blood separation process. In anotherembodiment, the filter is provided without the presence of chemical testreagents. For example, referring to an alternative embodiment of theinvention, as shown in FIG. 1, a multi-layer fructosamine test devicecontaining the blood separation matrix of the present invention incombination with a membrane can be used to determine the concentrationof fructosamine present in whole blood. Referring now to FIG. 1, afructosamine multi-layer test device 1 has a blood separation matrix 4and membrane 5, below which is the reagent layers, including a bufferlayer 6 and an indicator layer 7 as well as clear plastic window 8 forreading the test results on the indicator layer 7. A mesh layer 3, usedto press the multi-layers together, and the separation matrix 4 arecontained in a guard piece 11 and sealed with the membrane 5. Layers 6,7, and 8 are contained in opening 12 of well 13 which is on plasticsupport member 9. Pieces 11 and 13 containing the respective layers areultrasonically welded together 2.

Test reagents for determining the presence or concentration offructosamine such as the appropriate buffers and indicator reagents,including chromogenic dyes, or fluorescent reagents, are known in theart, for example, as described in U.S. patent application Ser. No.08/269,351, which is incorporated herein by reference.

The buffer layer 6 of the fructosamine test generally contains a bufferhaving a pH value of at least 9. Various known buffers can be containedin the buffer layer so long as the buffer provides sufficiently high pHsuch that the fructosamines are converted to their eneaminol form. Theeneaminol form of fructosamine is a chemically active reducing substancethat reacts with a suitable indicator capable of being reduced byfructosamine. To achieve this, the pH of the buffer should be at a pHvalue between about 9 and about 13 and, for optimum results, the pH isat a pH value of between 10 and 12. Examples of such buffers includepotassium hydrogen phosphate, sodium hydrogen phosphate, sodiumhydroxide, guanidinium salts, certain amino acids, and other suitablebuffers as are well known in the art, or combinations thereof.

The indicator layer 7 of the fructosamine test device contains anyindicator capable of being reduced by fructosamine such as certain dyes,including chromogenic dyes, or fluorescent reagents. Examples ofsuitable chromogenic dyes which change color based on the amount offructosamine present in a liquid sample include tetrazolium dyes such asNeotetrazolium chloride (NT), Tetranitroblue tetrazolium chloride(TNBT), Blue tetrazolium chloride (BT), Iodonitrotetrazoilum chloride,Nitroblue tetrazolium chloride (NBT), Nitro blue monotetrazoliumchloride, Thiazolyl blue tetrazolium bromide (MTT), Tetrazolium violet,2,3,5-Triphenyl-2-H-tetrazolium chloride, Thiocarbamyl nitro bluetetrazolium chloride (TCNBT), Tetrazolium XTT (XTT),2-2'-Benzothiazolyl-5-styryl-3-(4'-phthalhydrazidyl) tetrazoliumchloride (BSPT), Distyryl nitroblue tetrazolium chloride (DSNBT). Anexample of a suitable fluorescent reagents is 5-Cyano-2,3-ditolyltetrazolium chloride (CTC).

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE Fructosamine Test

This Example provides the preparation and testing of a multi-layerfructosamine test device using a whole blood separation matrix of thepresent invention. Because the presence of red blood cells normallyinterferes substantially with the analysis of fructosamine in wholeblood, this Example compares testing for fructosamine in a whole bloodsample using the present invention versus testing for fructosamine in aserum sample.

A. Blood Separation Layer

Mesh: A Tetko mesh #7-280/44 (Tetko, Inc. Rueschlikon, Switzerland) wasplaced in a detergent solution of 1% Pluronic (Pragmatics, Inc.) for 1minute. Excess detergent was removed and the mesh was dried by heatingat 60° C. for 10 minutes. Mesh was stored in desiccated plastic bagsuntil ready for use at which time a 3/16" circle of the mesh was placedin the fructosamine multi-layer test device.

Blood Separation Matrix: A solution of 10% mannitol and 1.25%hexadimethrine bromide in physiological saline (0.85% NaCl) wasimpregnated onto Sontara® #8007 (DuPont, Inc.) on an automatedimpregnation/drying unit (AFM Engineering, Santa Anna, Calif.). Thedrying temperature was 100° C. for approximately 10 minutes.

Membrane: An untreated BTS polysulfone membrane of 0.85 p/n pore size(Memtek, Inc.) was cut into a 3/16" for use as an additional filterbelow the blood separation matrix.

B. Reagent Layers:

Buffer Layer: A 1M solution of aqueous sodium phosphate (aqueous NaH₂PO₄) containing 1M guanidinium carbonate buffer was titrated with sodiumhydroxide (NaOH) to yield a 100 ml solution at pH 11. After addition of0.5% Surfactant 10G detergent (Pragmatics, Inc.) the mix was impregnatedonto Whatman 540 paper and dried for 10 minutes at 100° C.

Dye Layer: A 10 mM methanolic solution of nitroblue tetrazolium chloride(NBT) containing 200 μM N-ethylmethoxyphenazine ethylsulfate and 1%Gantrez® AN 119 was impregnated onto Whatman 54 paper and dried for 15minutes at 60° C.

The layers (3/16" circles) were assembled as follows:

Tetko Mesh (top)

Mannitol Containing Matrix

Polysulfone Membrane

Buffer Layer

Dye Layer (bottom)

The layers were held in place by an injection molded plastic part. Wholeblood and serum (15 μl) from the same donor were applied at the top andreaction rates were measured at the bottom as follows:

    ______________________________________                                                                 ΔK/S                                           Sample         Starting K/S                                                                            (1 to 2 min.)                                        ______________________________________                                        whole blood    0.214     0.184                                                serum          0.201     0.184                                                ______________________________________                                    

The results demonstrate that the reaction rate with the whole bloodsample is the same as that with the serum sample. These results indicatethat there was no interference from red blood cells and, therefore, thatthe whole blood separation of the present invention successfully removedthe red blood cells present in the whole blood sample.

EXAMPLE II Glucose Test

This Example demonstrates the preparation and testing of a rapid glucosetest containing a blood separation matrix of the present invention. TheExample tests for glucose in spiked whole blood samples.

A. Blood Separation Matrix:

A #8007 Sontara® impregnated with 10% mannitol and 1.25% hexadimethrinebromide was prepared as described above, except that the sugar alcoholand polymer were dissolved in water.

B. Filter Membrane and Chemical Reagent Layer: A sheet of 0.45 μmBiodyne A® nylon (Pall Corp.) was dipped into an aqueous solutioncontaining the following reagents:

    ______________________________________                                        0.30 M    Citrate Buffer                                                      1.25%     Gelatin 150 Bloom                                                   1106 U/ml Glucose Oxidase                                                      479 U/ml Horseradish Peroxidase                                              0.43%     4-Aminoantipyrine (AAP)                                             1.52%     N-ethyl-N-(2-hydroxyl-3-sulfopropyl)-m-toluidine                              Sodium Salt (TOOS)                                                  0.25%     Pluronic L64 (Poly(oxyethylene-co-oxypropylene)                               block polymer                                                       1%        Gantrez ® L139                                                  ______________________________________                                    

After dipping, the sheet was dried for 20 minutes at 50° C. The bloodseparation matrix was mounted on top of the nylon membrane containingthe test reagents and the two held together by adhesive. A drop each offour glucose spiked blood samples were applied to the first surface ofthe mannitol containing Sontara® matrix. The results are as follows:

    ______________________________________                                        Glucose Level  K/S                                                            (mg/dl)        (at 45 seconds)                                                ______________________________________                                         98            0.732                                                          180            0.986                                                          297            1.312                                                          450            1.747                                                          ______________________________________                                    

These results demonstrate that the color produced was proportional tothe glucose concentration in the blood sample and that a lineardose/response curve was accurately obtained, indicating goodperformance.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific examples detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

We claim:
 1. A process for separating plasma from a whole blood sample,comprising:(a) applying the whole blood sample to a first surface of apermeable non-glass fiber matrix containing (1) a C₃ to C₂₀ polyhydroxyalcohol capable of clumping red blood cells and (2) a polycationicpolymer, said matrix being porous to red blood cells in the absence ofsaid polyhydroxy alcohol; (b) flowing said whole blood sample underconditions which allow said sample to flow vertically toward a secondsurface of said matrix, said first and second surfaces being on oppositesides of said matrix; and (c) receiving plasma separated from said wholeblood sample at said second surface.
 2. The process of claim 1, whereinthe polycationic polymer is hexadimethrine bromide.
 3. The process ofclaim 1, wherein said matrix is supported by a permeable filtermaterial.
 4. The process of claim 3, wherein the permeable filtermaterial is a reagent layer containing at least one chemical reagent fordetermining the presence of an analyte in the plasma.
 5. The process ofclaim 4, wherein said chemical reagent provides a color determination.6. The process of claim 4, wherein determining the presence of ananalyte is a quantitative determination.
 7. The process of claim 3,wherein the permeable filter material is selected from the groupconsisting of a nylon, a cellulose acetate, a polysulfone, a syntheticfiber, and a polycarbonate.
 8. The process of claim 1, wherein thepolyhydroxy alcohol is impregnated in the matrix.
 9. The process ofclaim 1, wherein the matrix is selected from the group consisting of apolyester, a nylon, a rayon, and a cotton.
 10. The process of claim 1,wherein the matrix is a non-woven, non-absorbent polyester.
 11. Theprocess of claim 10, wherein the polyester is a poly(paraphenyleneterephthalate).
 12. The process of claim 1, wherein the polyhydroxyalcohol is present in a concentration of about 10%.
 13. A device forseparating plasma from a whole blood sample, comprising a permeablenon-glass fiber matrix containing (1) a C₃ to C₂₀ polyhydroxy alcoholcapable of clumping red blood cells and (2) a polycationic polymer, saidmatrix being porous to red blood cells in the absence of saidpolyhydroxy alcohol, said matrix further having a first surface and asecond surface which are on opposite sides of said matrix, wherein thewhole blood sample vertically flows from said first surface toward saidsecond surface and wherein plasma separated from a whole blood samplebecomes available at said second surface.
 14. The device of claim 13,wherein the polycationic polymer is hexadimethrine bromide.
 15. Thedevice of claim 13, wherein the matrix is supported by a permeablefilter material.
 16. The device of claim 15, wherein the permeablefilter material is a reagent layer containing at least one chemicalreagent for determining the presence of an analyte in the plasma. 17.The device of claim 15, wherein the permeable filter material isselected from the group consisting of a nylon, a cellulose acetate, apolysulfone, a synthetic fiber, and a polycarbonate.
 18. The device ofclaim 13, wherein the polyhydroxy alcohol is impregnated in the matrix.19. The device of claim 13, wherein the matrix is selected from thegroup consisting of a polyester, a nylon, a rayon, and a cotton.
 20. Thedevice of claim 13, wherein the matrix is a non-woven, non-absorbentpolyester.
 21. The device of claim 20, wherein the polyester is apoly(paraphenylene terephthalate).
 22. The device of claim 13, whereinthe polyhydroxy alcohol is present in a concentration of about 10%. 23.A device for separating plasma from a whole blood sample, comprising:(a)a non-woven, non-absorbent polyester matrix impregnated with mannitoland hexadimethrine bromide, said matrix being porous to red blood cellsin the absence of said mannitol and said matrix further having a firstsurface and a second surface which are on opposite sides of said matrix,wherein the whole blood sample vertically flows from said first surfacetoward said second surface and wherein plasma separated from the wholeblood sample becomes available at said second surface; and (b) a nylonmembrane supporting said matrix.
 24. The device of claim 23, whereinmannitol is present in a concentration of about 10%.
 25. A process forseparating plasma from a whole blood sample, comprising:(a) applying thewhole blood sample to a first surface of a permeable non-glass fibermatrix containing (1) a C₃ to C₂₀ polyhydroxy alcohol capable ofclumping red blood cells and (2) a polycationic polymer, said matrixbeing porous to red blood cells in the absence of said polyhydroxyalcohol; (b) flowing said whole blood sample under conditions whichallow said sample to flow directionally toward a second surface of saidmatrix; and (c) receiving plasma separated from said whole blood sampleat said second surface.
 26. The process of claim 25, wherein thepolyhydroxy alcohol is mannitol and further wherein the polycationicpolymer is hexadimethrine bromide.
 27. The process of claim 25, whereinthe polyhydroxy alcohol is present in a concentration of about 10%. 28.A device for separating plasma from a whole blood sample, comprising apermeable non-glass fiber matrix containing (1) a C₃ to C₂₀ polyhydroxyalcohol capable of clumping red blood cells and (2) a polycationicpolymer, said matrix being porous to red blood cells in the absence ofsaid polyhydroxy alcohol, said matrix further having a first surface anda second surface, wherein the whole blood sample is applied to saidfirst surface and flows directionally toward said second surface andfurther wherein plasma separated from a whole blood sample becomesavailable at said second surface.
 29. The device of claim 28, whereinthe polyhydroxy alcohol is mannitol and further wherein the polycationicpolymer is hexadimethrine bromide.
 30. The process of claim 28, whereinthe polyhydroxy alcohol is present in a concentration of about 10%.